1. Field of the Invention
The present invention relates generally to infrared (IR) spectroscopy systems and methods for in-vitro analysis of biological cells and/or microorganisms. More particularly, the systems and methods of the present invention allow specific and quick analysis of degree of differentiation, cell types, donor individuals, culture conditions, purity, lack of natural characteristic, or additional characteristics in comparison to natural characteristic.
2. Background Art
In the past decade application of different suitable medical and non-medical techniques have greatly advanced areas of clinical study. It is well known that the ease of medical treatment depends greatly on the speed of a diagnosis. Thus, the sensitivity and precise discriminatory nature of early diagnostic methods are very important in the effective medical treatment of patients. The potential of techniques for screening and disease diagnosis in clinical settings, as well as in the medical area regarding transplants, has been already investigated by various research groups. A number of these techniques have been applied in recent years to study different biological objects such as biological fluids and tissues. Many of these techniques are aimed specifically to identify a certain pathological conditions or to determine, differentiate and typify different biological cells or microorganisms.
Until now, the most conventional method for controlling, identifying, typifying, quantifying, classifying and monitoring biological objects, such as different types of biological cells and microorganisms as well as starting materials and the finished products used for example in the transplantation medicine, is the Polymerase Chain Reaction (PCR) technique. For an emerging gene-based identification method, genus-specific or species-specific PCR primers or nucleotide probes are applied to an object of interest. However, there are some limitations to the use of PCR such as (i) greater expense than starting PCR as twice as much enzyme and reagents are used, (ii) extra manipulations and length of the assay as cycling blocks cannot be programmed for all cycles from the start, and (iii) applies only to short DNA fragments. Further, such determinations are very time-consuming and require sophisticated apparatuses to carry out the different tests.
A further concept is the use of cell type specific markers. In this respect it is very difficult to find cell type specific markers addressed only to one specific cell type. This concept has the problem that only quantities in expression can be determined but no answer is given whether a different type of cell is present or not.
Other standard methods to read out biological systems, in particular cells or microorganisms, are for example fluorescent and chemiluminescent-imaging techniques as well as NMR spectroscopy:
(i) Fluorescent and chemiluminescent-imaging methods require labeling of a component by a marker and usually only detect the concentration of the marker molecule.
(ii) Although solution NMR-spectroscopy provides a detailed image of the structure of a bio-object, the NMR method is much more complex. Furthermore, for the NMR analysis the large quantities of the samples are needed which have to be further labeled by isotopes of carbon (13C), nitrogen (15N), oxygen (17O), and hydrogen (2H).
Infrared (IR) spectroscopy is a technique routinely used by chemists, biochemists, and material scientists as a standard analyzing method. The observed spectroscopic signals are due to the absorption of infrared radiation that is specific to functional groups of the molecule. These absorption frequencies are associated with the vibrational motions of the nuclei of a functional group and show distinct changes when the chemical environment of the functional group is modified. Infrared spectroscopy essentially provides a molecular fingerprint and has thus always the potential as a diagnostic and monitoring tool in biology and medicine. Infrared spectra contain a wealth of information on the molecule, in particular they are used for the identification and quantification of molecular species, the interactions between neighboring molecules, their state of hydration, their overall shape, etc. Infrared spectra can be used as a sensitive marker of structural changes of cells and of reorganization occurring in cells. The diagnostic potential of infrared spectroscopy is being realized in many medical research programs based on the fundamental premise that in any pathologic process, the chemical change must precede the morphological or symptomatic manifestation. Organic applications of IR spectroscopy are almost entirely concerned with frequencies in the range of 400-4000 cm−1 (mid-infrared). Frequencies lower than 400 cm−1 are called far-infrared and those greater than 4000 cm−1 are called near-infrared.
Thus, there are several important advantages in using this technique: results are obtained relatively quickly with less labor input than many other techniques. The use of IR spectroscopy may provide more precise information on the exact nature of, for example, a disease based on sampling of a biological fluid or tissue. The method also allows to monitor the dynamics of the characteristic change, which is important in determining the exact stage of a biological object, such as cells or microorganisms, or of a disease.
Erukhimovitch et al. (Photochemistry and Photobiology, 2002, 76(4), 446-451) discloses a method for the diagnosis and characterization of cell and tissue pathology. In particular, Erukhimovitch et al. discloses the use of Fourier Transform Infrared (FT-IR) microspectroscopy instead of conventional FT-IR spectroscopy as having advantages in the diagnostic of malignancies. For this purpose FT-IR microspectra of two types of retrovirus-transformed malignant cells were compared with those of non-transformed primary cells and with those of two types of cell lines. Erukhimovitch et al. suggests using, for example, a band corresponding to the PO2− symmetric stretching mode to control the levels of phosphate and other metabolites. Erukhimovitch et al. discloses the use of FT-IR microspectroscopy for the investigation of the metabolism in mouse and human cells by comparing the FT-IR microspectra of two types of retrovirus-transformed malignant cells with those of non-transformed primary cells and with those of two types of cell lines. Thus, Erukhimovitch et al. is directed to study of a metabolism of cells which can be considered as an instantaneous image of the cell status and can be further used for classification of the cell metabolic activity and cell metabolic status.
However, the principal problem in FT-IR microspectroscopy is the presence of scattered light due to diffraction which limits the spatial resolution by increasing the noise. Diffraction is significant when the aperture dimensions approach the wavelength of the IR radiation. The main effect of diffraction is that at small aperture sizes, light spreads outside the specified area into the surrounding region. As higher spatial resolution is sought, the problem increases, as the apertures are smaller, ultimately leading to loss of spectral quality and photometric accuracy. Therefore, the spectrum obtained does not always correspond exactly to the area that is observed visually and defined by the remote aperture. This is especially true when the sample dimensions are very small. Furthermore, any time that measurements are made when the size of the sample that is being observed is equal to, or slightly greater than, the diffraction limit, some radiation is transmitted to the detector from outside the region that was selected by the remote aperture.
Oberreuter et al. (Letters in Applied Microbiology, 2000, 30, 85-89) discloses FT-IR determination of the ratios of different microorganisms in mixtures comprising a two-component food-associated yeast system and a two-component yoghurt lactic acid bacteria system. In particular, Oberreuter et al. suggests to use FT-IR spectroscopy for the evaluation of the calibration curves of single components and a mixture comprising Saccharomyces cerevisiae and Hanseni-aspora uvarum as well as a mixture comprising Lactobacillus acidophilus and Streptococcus salivarius, which can be further used for the quantification of these microorganisms in a mixture. Oberreuter et al. uses FT-IR spectroscopy for differentiation between individual species present in a mixed population.
Methods for the determination, especially typification and status check of well or fully differentiated mammalian cells are required especially in the field of transplantation medicine. For example, it is possible to produce replacement tissue by taking intact cells of the suitable tissue type from the transplant recipient, cultivating them in-vitro and re-introducing them into the patient after the necessary cell count has been reached. This can be effected either in the form of solutions or cultivated tissue portions or by cultivating the cells on a matrix (which preferably may be absorbed biologically) and re-implanting them together with the matrix. For example, suitable methods, matrices and cultivation media are described in the German applications 101 62 205.8, 101 62 960.5, 102 20 368.7, 102 22 896.5 (corresponds to AU 2003240618 A1) and the literature cited therein.
DE 103 26 966 A1 and its equivalent US 2006/0008795 A1 refers to a method for the in-vitro determination, especially typification, of well of fully differentiated mammal cells using Infrared Spectroscopy, especially their Fourier transformation (FT-IR). In particular, DE 103 26 966 A1 discloses the use of FT-IR spectroscopy for differentiation between well and fully differentiated cells of one specific species.
IR spectroscopy methods suggested in the prior art have similar limitations: they allow for selective determination or identification only of some biological objects such as bacteria, yeast or mammal cells.
Especially in the field of tissue engineering, transplant medicine as well as in questions of regulatory affairs and drug safety of in-vitro cultured cells and/or microorganisms there is a need to assure the quality, purity and origin of biological cells and/or microorganisms.
There is a need for a universal technique that would allow for a quick, broad examination of various biological objects such as biological cells and/or microorganisms as well as for distinguishing their characteristics.